The transcriptional complexity of cancer: a breast cancer experience

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  • THE TRANSCRIPTIONAL COMPLEXITY OFCANCER: A BREAST CANCER EXPERIENCE

    Ileana Zucchi1

    1. Istituto di Tecnologie Biomediche, Consiglio Nazionale delle

    Ricerche, Milan, Italy

    The genetic nature of cancer and the cancer transcriptome havebeen explored for more than one decade, but only recently havethe interactions of the epigenomic and genomic components incancer cells become apparent in tumor development. Theseinsights have largely been due to the availability of new technol-ogies, such as affordable massively parallel sequencing, ChiP-Seq,and non-coding microRNA array-based expression profiling, thatprovide a combined framework for exploring the transcriptionalcomplexity of cancer. For understanding the natural history ofhuman tumor development, deep sequencing was used to detectrare and novel non-coding transcripts and combined with micro-RNA-array technologies to investigate the composition and regu-lation of the cancer transcriptome by miRNAs and non-codinggene fusion transcripts. To optimize the detection of rare andnovel transcripts, a cDNA library normalization step was intro-

    WHOLE GENOME PROFILING ON FRESHLYFROZEN AND MATCHING ARCHIVED ANDFRESHLY PREPARED FORMALIN-FIXED

    61Abstracts / Cancer Genetics and Cytogenetics 203 (2010) 44e65duced to diminish the representation of highly expressed tran-scripts. We obtained and analyzed over 132,000 high-confidencedeep sequencing reads from primary human lobular breast cancertissue specimen. We detected thousands of novel non-coding tran-scripts and unique transcriptional events, and a select numberwere subsequently validated in additional primary human breastcancer samples. We are currently exploring the mechanism bywhich the identified unconventional transcripts such as non-codingRNAs, miRNAs, somatic gene fusions and deletions may influ-ence mRNA transcript levels and the chromatin remodeling stateof tumor cells. Our results demonstrate that by combining conven-tional and recent technologies for transcriptomic analysis we canprovide insight into how interactions of epigenomic and transcrip-tomic components can contribute to human tumor developmentand offer promising strategies in the search for potential targetsfor therapeutic intervention.PARAFFIN-EMBEDDED TISSUES

    Vanja de Weerd1, Anieta M. Sieuwerts1, Raquel Ramrez-Moreno1,2,

    Renee Foekens1, Mieke Timmermans1, Marcel Smid1, John A. Foekens1,

    John W.M. Martens1

    1. Department of Medical Oncology, Erasmus MC/Daniel den Hoed

    Cancer Center/Josephine Nefkens Institute, Rotterdam, The

    Netherlands

    2. Department of Biochemistry and Molecular Biology Physiology,

    Genetic and Immunology, University of Las Palmas de Gran

    Canaria, Canary Islands Cancer Research Institute, Canary Islands,

    Spain

    Formalin fixation of human tissue and subsequent embedding inparaffin has been a routine method of collecting and preservingsurgical specimens for decades. These formalin-fixed, paraffin-embedded (FFPE) tissues represent a potential extremely valuableresource for molecular studies. The analysis of nucleic acids ofFFPE specimens has proven to be technically challenging, becausenucleic acids are typically degraded and modified. This issuebecomes increasingly problematic with aging of specimens.WG-DASL (Whole GenomeecDNA-mediated Annealing, Selec-tion, extension and Ligation) is a bead-based method developedby Illumina to analyze the whole genome (20,000 genes).Contrary to the Direct Hybridization method, also developed byIllumina (40,000 genes) for fresh frozen (FF) material, the WG-DASL even works with highly degraded RNA samples. In thisstudy we set out to compare the RNA quality from archived(1980e2000) and more recently (2009) embedded FFPE tissueswith FF material from the same tissues and its performance inWG gene expression profiling. FFPE- and FF-RNA was obtainedfrom primary breast tumor specimens. FFPE-RNA was isolatedwith the Roche High Pure RNA Paraffin kit, FF-RNA withRNA-Bee. RNA quality was checked by gel-electrophoresis andits performance in real-time reverse-transcriptase PCR. Next tothese standard quality control measures, 1 mg FFPE-RNA and0.5 mg FF-RNA was used for WG-DASL profiling. Data qualitycontrol was assessed with GenomeStudio (Illumina) and the geneexpression profiles of FFPE samples were compared with thoseobtained from matched FF samples profiled by Direct Hybridiza-tion (Illumina) and on U133a chips (Affymetrix). Differences inRNA quality were observed when comparing archived FFPE spec-imens with recently embedded specimens. Gene expression profilesgenerated by WG-DASL were reproducible for duplicate archivedFFPE specimens (Spearman R-values [Rs] range from 0.92 to0.98). The average call rate for FF-RNA was 46.1%, for FFPE-RNA 38.6%. WG-DASL-profiles were comparable to profilesgenerated by Direct Hybridization for FF samples (Rs 5 0.69,n5 11). For FF samples, profiles generated by Direct Hybridizationmethod were comparable to profiles generated on the Affymetrixplatform (Rs 5 0.60, n 5 9). Despite clear differences in RNAquality, our data show that gene expression profiles generated byWG-DASL from FFPE-RNA are reproducible and that the datacan be compared with FF-RNA gene expression profiles.

    The transcriptional complexity of cancer: a breast cancer experienceWhole genome profiling on freshly frozen and matching archived and freshly prepared formalin-fixed paraffin-embedded tissues

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